Influenza virus attachment to host cells is mediated by specific interactions of the viral envelope protein hemagglutinin (HA) with sialylated carbohydrate chains of cell surface glycoproteins and glycolipids (for reviews, Suzuki, 1994; Herrler et al., 1995; Paulson, 1985; Wiley and Skehel, 1987). Natural sialylglycoproteins and gangliosides exhibit significant structural diversity, and different receptors are probably utilized by the viruses in different host tissues. It has been shown, that influenza A viruses isolated from avian species preferentially bind to NeuAcα3Gal-terminated sugar chains, while closely related human viruses reveal a higher binding affinity towards the NeuAcα6Gal-terminated structures (Paulson, 1985; Suzuki, 1994; Connor et al., 1994; Matrosovich et al., 1997; Gambaryan et al, 1997). Additional influential features for binding are inner parts of saccharide chains (Gambaryan et al., 1995; Matrosovich et al., 1997; Rogers and Paulson, 1983; Suzuki et al., 1987; Suzuki et al., 1992; Eisen et al., 1997), polyvalency of receptor saccharides (Pritchett and Paulson, 1989; Matrosovich, 1989; Mammen et al., 1995), spatial arrangement of sialyloligosaccharides in receptor glycoproteins (Pritchett and Paulson, 1989), or glycosylation of the viral hemagglutinins (Gambaryan et al., 1998; Ohuchi et al., 1997). Detailed molecular mechanisms of these effects and the importance of a variation in fine structure of sialylated receptors for the virulence and pathogenicity of individual viral strains are not known. For example virulence of the 1918 influenza pandemic still remains unexplained (Laver et al., 1999), and the actual strains have not been assayed for receptor specificity.
Studies on the structural characterization of biological receptors for human influenza viruses are hampered by the limited availability of the human respiratory tract tissues. However, characterization of the binding molecules from other human tissues may permit further specification of the receptor binding epitopes. Human leukocytes represent an attractive experimental model because they contain a series of gangliosides with high binding affinity for the virus. Binding species were detected in human leukocytes among common gangliosides (Müthing et al. 1993; Müthing, 1996). In contrast to the present invention these were described to contain α3-sialylated sialyl-Lewis x and VIM-2 sequences. The receptor activity was also observed among highly complex glycolipid fractions, polyglycosylceramides (Matrosovich et al., 1996). NeuAcα6-containing glycolipids of human leukocytes with more than two lactosamine units in the core chain have not yet been characterized by other laboratories (Müthing et al., 1993; Müthing et al. 1996; Müthing, 1996; Stroud et al., 1995; Stroud et al., 1996). They occur in human white cells in very small amounts and their existence has so far been neglected. However, these minor species may be of biological importance for in vivo events during influenza infections and may explain virulence variations between strains (Laver et al., 1999).
Binding of influenza viruses to sialic acid-containing neutrophil receptor(s) depresses bactericidal activity of neutrophils (Abramson and Mills, 1988; Cassidy et al., 1989; Daigneault et al., 1992; Abramson and Hudnor, 1995) and stimulates apoptosis of these cells by a yet undefined mechanism (Colamussi et al., 1999). This virus-mediated neutrophil dysfunction is a likely contributor to the development of secondary bacterial infections, which are the main cause of morbidity and mortality during influenza epidemics.
Several studies describe sialylated di- to heptasaccharide binding structures for human influenza viruses e.g. (Gambaryan et al., 1995; Matrosovich et al., 1997; Rogers and Paulson, 1983; Suzuki et al., 1992; Eisen et al., 1997). These have low affinities to the viruses and the studies do not describe larger saccharide receptors which have the high natural binding affinity. The divalent NeuNAcα6Galβ4GlcNAc-saccharides constructed chemically on β-galactosides are described in Sabesan, S. et al. -92 and in U.S. Pat. Nos. 5,254,676 and 5,220,008 and have only modestly better affinities than corresponding monosialocompounds even when linked on bovine serum albumin in multivalent form.
Several synthetic polymeric influenza inhibitors containing sialic acid (Mammen et al., 1995) or sialylated lactose/N-acetyllactosamine have been described (Gambaryan et al, 1997). The specific binding of human influenza virus to NeuNAcα6Galβ4GlcNAc-epitopes (Gambaryan et al, 1997) and the presence of NeuNAcα6Gal on the surface of human-ciliated tracheal epithelium (Baum and Paulson, 1990) were suggested to constitute an essential part of the biologically relevant cellular receptor for the viruses (Gambaryan et al, 1997).
A polylactosamine containing and α6-sialylated inhibitor of human influenza viruses has been described. This molecule is produced from a cryptically I-active (anti-I-antibodies are known to recognize polylactosamine and non-polylactosamine structures) glycoprotein 2 of bovine erythrocytes by removing sialic acid residues by sialidase treatment and by enzymatic resialylation by α3- or α6-sialyltransferases (Suzuki et al., 1987). The semisynthetic bovine protein is not a natural receptor structure of human viruses. It is hardly useful as a therapeutic inhibitor of human influenza viruses, because it contains a protein structure and a substantial amount of Galα3Galβ4GlcNAc-xeno-antigenic structures (Suzuki et al, 1985). The Galα3Galβ4GlcNAc-antigen is present in many mammalian species but not produced by human tissues, the structure is highly antigenic in human, and there are naturally large amounts of antibodies against the structure in humans. Foreign protein structures are known to be potential antigens and allergens for humans.
The authors (Suzuki et al., 1987) discuss that inner I-active neolacto-series type II sugar chains may also be important as a common part of the receptor determinant toward the hemagglutinin of human influenza viruses A and B. Unfortunately their saccharide material is very heterogenous and the possible sialylated I-active components formed were not characterized chemically and no specific epitopes were assigned as virus binding structures. According to the data of FIG. 3 corresponding to oligosaccharides labelled at the reducing end, the large I-polylactosamines are actually very minor species in the glycoprotein II and smaller O-glycans are major species in molar amount, the smaller O-glycans without branched polylactosamime are also recognized by at least one anti-I-antibody used to determine the saccharides of the protein (Suzuki et al., 1985). In another study the same authors show that α3-sialylated and α3-galactosylated branched polylactosamine glycolipid (also an I-antigenic structure) was shown to be a binding compound for influenza virus (Suzuki et al., 1986).